General. Unless otherwise noted, all starting materials were obtained from commercial
suppliers and were used without further purification.
Bacterial Growth and Crude Nucleic Acid Isolation. E. coli TOP10 (Invitrogen) was cultured
to OD600 = 0.7-0.8 at 37ºC in 2 L LB broth Miller (EMD Bioscience). S. venezuelae ATCC
#10595 was cultured to OD600 = 0.7-0.8 at 30 ºC in 2 L MYME media (100 g/L sucrose, 10 g/L
maltose, 5 g/L peptone, 3 g/L yeast extract, 3 g/L malt extract).140 All subsequent manipulations of cells were carried out on ice or at 4 °C. The bacteria were centrifuged (10 min at 6,750 x g), resuspended in lysis buffer (1% SDS, 2 mM EDTA, 32 mM NaOAc, pH 4.5), and vortexed vigorously. After incubation on ice for 15 min, the lysate was cleared by centrifugation (10 min at 10,000 x g), extracted with acid-phenol chloroform (Ambion) until the organic-aqueous interface was clear, and the aqueous layer was washed once with chloroform. An equal volume of isopropanol was added to the resulting aqueous extract and the mixture was incubated on crushed dry ice 20-30 min prior to centrifugation (20 min, 15,000 x g). The resulting pellet was dissolved in 50 mM NH4OAc, pH 4.5, and subjected to size-exclusion chromatography using
NAP5 columns (GE Healthcare). The macromolecular fraction was treated with 0.04 U/µL TURBO DNase (Ambion) at 25 ºC for 30 min and then with 0.06 U/µL proteinase K (New England Biolabs) at 25 °C for 30 min. The resulting solution was extracted with acid-phenol chloroform (Ambion) twice, washed with chloroform, and again subjected to size-exclusion
chromatography using NAP5 columns. The macromolecular fraction was divided into aliquots, lyophilized, and stored as a dry powder at -80 ºC.
Nuclease Digestion. The cleavage condition sample was prepared by incubating 500 µg of E.
coli RNA or 500 µg of S. venezuelae RNA with 10 U nuclease P1 (Sigma-Aldrich) in 200 µL of 50 mM NH4OAc, pH 4.5 at 37 ºC for 40 min. The control condition samples was prepared by
incubating 500 µg aliquot of E. coli RNA or S. venezuelae RNA with 10 U heat-inactivated nuclease P1 (95 ºC for one hour) in 200 µL of 50 mM NH4OAc, pH 4.5 at 37 ºC for 40 min. The
digestion products were purified by size-exclusion chromatography (NAP5) and the small- molecule fraction was retained. The nuclease P1 digestion with H218O (Cambridge Isotope
Laboratories) was performed as described above except in buffer with a final composition containing 86% H218O and 14% H216O.
LC/MS Data Collection and Analysis. LC/MS was performed using a Waters Aquity UPLC
Q-TOF Premier instrument with an Aquity UPLC BEH C18 column (1.7 µm, 2.1 mm x 100 mm, Waters). Mobile phase A was 0.1% aqueous ammonium formate, and mobile phase B was 100% methanol. The flow rate was a constant 0.300 mL/min and the mobile phase composition was as follows: 0% B for 3 min; linear increase over 17 min to 100% B; maintain at 100% B for 2 min before returning linearly to 0 % B over 1 min. Electrospray ionization (ESI) was used, with a capillary voltage of 3.5 kV, sampling cone voltage of 40.0 kV, and collision voltage of 1.0 eV. The drying gas temperature was 300 ºC, the drying gas flow rate was 800 L/hour, the source temperature was 150 ºC, and the detector was operated in negative ion mode. To observe the NAD parent ion ([M-H]- m/z = 662.1018), the capillary voltage was 2.75 kV and the sampling cone voltage was 20.0 kV. For each sample, 15 µL of the redissolved lyophilized material was
61 injected. Ions with cleavage condition average integrated ion intensities below 50 ion counts were not considered for further analysis. The XCMS program109 quantified the area under detected ion abundance peaks as it stepped through each ion chromatogram; the step size was set to 0.050 Da. Integrated ion abundances were averaged among replicates, and the enrichment values reported were the ratios of these average ion intensities between active nuclease conditions and heat-inactivated nuclease (control) conditions.
MS/MS Fragmentation Analysis. MS/MS experiments were performed using the same
instrument, LC gradient, and MS parameters described above. The collision voltage was varied empirically from 20.0-30.0 2V.
Isotope Labeling of S. venezuelae RNA. S. venezuelae ATCC #10595 was cultured to OD600 =
0.7-0.8 at 30 °C in minimal media (4.3 g/L NaH2PO4, 6.1 g/L K2HPO4, 2.0 g/L NaCl, 2.0 mg/L
FeSO4·7H2O, 2.0 mg/L MnCl2·4H2O, 2.0 mg/L ZnSO4·7H2O, 2.0 mg/L CaCl2, 0.6 g/L
MgSO4·7H2O, 10.0 g/L glucose, and 2.0 g/L (NH4)2SO4)140. For 13C labeling, 13C-glucose (99% 13C, Cambridge Isotope Laboratories) was used as the sole carbon source. For 15N labeling, 15N-
ammonium sulfate (99% 15N, Cambridge Isotope Laboratories) was used as the sole nitrogen source. RNA isolation, nuclease P1 digestion, and LC/MS analysis was performed as described above.
NAD Spiking Into Cell Lysate. E. coli TOP10 cell lysate was prepared as described above and
divided into three equal aliquots. NAD and NADH (Sigma-Aldrich) were added to the cell lysate as follows: aliquot 1: no added NAD or NADH; aliquot 2: 500 nmol of NAD; aliquot 3:
500 nmol of NADH. Based on our standard curve analysis 500 nmol of NADH or 500 nmol of NAD theoretically represents 4,000,000 ion counts; in practice, this amount of NAD or NADH saturates the detector. Cell lysates were processed and analyzed as described above.
RNase A Digestion. Total RNA prepared as described above was subjected to digestion with 1
µg RNase A (Ambion) in 200 µL of 50 mM NH4OAc, pH 4.5, at 37 ºC for 20 min). After
digestion, the sample was subjected to size-exclusion chromatography (NAP5, GE Healthcare) and the small-molecule fraction was lyophilized. The lyophilized product was redissolved in 20 µL of 0.1 % aqueous ammonium formate and analyzed by LC/MS as described above.
Number of NAD-RNA(s) per E. coli cell. To establish a standard curve of MS ion counts per
pmol NAD, 5, 25, 50 and 100 pmols of authentic NAD (Sigma-Aldrich) were analyzed by initial and mild ionization conditions as described above. The NAD signal from 500 µg of total E. coli RNA was compared to the standard curves to determine the number of pmols of cellular NAD per µg RNA. The molecules of NAD-RNA per cell was then calculated based on 59x10-15 g
RNA per E. coli cell.141
Authentic NAD and NADH Carried Through Mock RNA Isolation. To determine if cellular
NADH-RNA could be detected by our nuclease based method, 50 pmol of NAD and NADH (Sigma-Aldrich) in 500 µL of 50 mM NH4OAc, pH 4.5 were incubated on ice for 15 min, and
then at 4 °C for 10 min. The samples were moved back to ice for 5 min and then placed at 4 °C for 10 min. This procedure was repeated three times, corresponding to the number of acid- phenol chloroform and chloroform extractions during the crude nucleic acid isolation. The NAD
63 and NADH samples were then placed on crushed dry ice for 20-30 min prior to incubation at 4 °C for 20 min, mimicking the isopropanol precipitation of cellular RNA. While the crude nucleic acids were subjected to size-exclusion chromatography, the NAD and NADH samples were exposed to air and incubated at 25 °C for 10-15 min. The samples were then incubated at 25 °C for 1 h, and lyophilized. The dry powder was redissolved in 200 µL of 50 mM NH4OAc,
pH 4.5 and incubated at 37 ºC for 40 min. The NAD and NADH samples were exposed to air and incubated at 25 ºC for 15-20 min. The samples were lyophilized and stored as a dry powder at -20 ºC.
Preparation of Authentic 5’-NAD-RNA. 5’-NAD-RNA was prepared as previously
described.135 A DNA template containing the T7 class II promoter (Φ2.5) and encoding a 228- base RNA was prepared by PCR. Transcription was carried out in 1x New England Biolabs (NEB) RNA pol buffer supplemented with 1 mM each NTP, 1 mM NADH (Aldrich), 0.01% Triton X-100, 5 mM DTT, 0.2 U/µl RNase inhibitor (NEB), 0.2 µM DNA template, and 5 U/µL T7 RNA polymerase (NEB). The reactions were incubated at 37 °C for 2 hrs before the product was isolated by precipitation with ethanol, dissolved in TURBO-DNase buffer (Ambion), and treated with TURBO-DNase (0.04 U/µL final concentration, 37 °C, 30 min). The DNase-treated RNA was precipitated with ethanol, redissolved in water, and purified by size-exclusion
chromatography (NAP5, GE Healthcare). The precipitated NAD-RNA product following DNase treatment was dissolved in water, purified by silica column (Qiagen RNeasy), quantified by A260,
digested with nuclease P1, and analyzed by LC/MS as described above in order to estimate the fraction of the resulting RNA strands that contained the NAD modification. We observed that ~3% of the transcripts generated by this procedure were linked to NAD.
In Vitro Transcription. In vitro transcription reactions contained E. coli RNA polymerase (Epicentre Biotechnologies) in 50 mM Tris-HCl, pH 7.5; 150 mM KCl, 10 mM MgCl2, 0.01%
Triton X-100 supplemented with NTPs (0.5 mM each), DTT (10 mM), and NADH (0.5 mM or 5.0 mM). The plasmid or genomic DNA template was added to a final concentration of 0.02 µg/µL and E. coli RNA polymerase was added to a final concentration of 0.04 U/µL. The
reactions were incubated at 37 °C for 22 hrs before the product was isolated by precipitation with ethanol, dissolved in TURBO-DNase buffer (Ambion), and treated with TURBO-DNase
(Ambion) at a final concentration of 0.04 U/µL for 30 minutes at 37 °C. The RNA was purified by RNeasy spin-column (Qiagen) to remove free NTPs and NADH. The resulting RNA was digested with nuclease P1 and analyzed by LC/MS as described above.
The first template was a modified pUC19 plasmid, which encoded an adenosine at the +1 position of each of its four predicted transcripts. An in vitro transcription reaction containing 0.5 mM of each NTP and either 0.5 mM or 5 mM of NADH yielded 222 µg or 174 µg of RNA, respectively. The second template used was E. coli genomic DNA. In vitro transcription in the presence of either 0.5 mM or 5 mM of NADH yielded 78 µg or 70 µg of RNA. Once again, this material contained no detectable NAD or NADH after nuclease P1 digestion.
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Chapter Three:
The Characterization of NAD-Linked RNA
Ye Grace Chen, Matt Edwards, David R Liu
Matt Edwards performed the alternate computational processing of Illumina Hi-Seq results. Ye Grace Chen conducted and analyzed all of the other experiments described.
69
3.1 Introduction
We previously described the development of a nuclease-based screen that revealed NAD- linked RNA. Further characterization of NAD-linked RNA, including the size distribution and sequence, may elucidate its biological function. A more comprehensive understanding can add to the known functional diversity of RNA and contribute to a greater insight of the RNA world. In this chapter, we will describe the progress toward the characterization of NAD-linked RNA.